METHOD FOR OBTAINING ENDOTHELIAL CELLS FROM PLURIPOTENT STEM CELLS

Abstract

The invention relates to a method for obtaining endothelial cells from human pluripotent stem cells.

Claims

1. A method for differentiating pluripotent stem cells into endothelial cells, comprising the steps of: a) on D0, the pluripotent stem cells are dissociated, seeded at a density of 40 000 to 60 000 cells/cm.sup.2 on a matrix and cultured in the presence of a medium suitable for the culture of pluripotent cells, further comprising fibroblast growth factor 2 (FGF2) and a ROCK inhibitor; b) on D1, the medium is replaced with a medium suitable for mesoderm induction, further comprising a GSK3 (Glycogen Synthase Kinase 3) inhibitor and BMP4 (Bone Morphogenetic Protein 4); c) on D4, the medium is replaced with a medium suitable for the culture of endothelial cells, further comprising VEGF (Vascular Endothelial Growth Factor) and forskolin; d) on D6, the cells are dissociated and selected for expression of the CD144 marker.

2. The method according to claim 1, wherein the matrix is the L7 matrix sold by Lonza under the reference FP-5020.

3. The method according to claim 1, wherein the FGF2 is used at a concentration from 5 to 20 ng/ml in step a).

4. The method according to claim 1, wherein the GSK3 inhibitor is Chir99021 in step b).

5. The method according to claim 1, wherein the BMP4 is used at a concentration between 15 and 50 ng/ml in step b).

6. The method according to claim 1, wherein the VEGF is used at a concentration between 100 and 300 ng/ml in step c).

7. The method according to claim 1, wherein the forskolin is used at a concentration between 1 and 3 μM in step c).

8. The method according to claim 1, wherein the pluripotent stem cells are human induced pluripotent stem cells.

9. An endothelial cell population obtained by the method as defined in claim 1.

10. A method for producing a dermal tissue, comprising obtaining endothelial cells by the method of claim 1, and producing the dermal tissue from the endothelial cells.

11. The method according to claim 1, wherein the FGF2 is used at a concentration of approximately 10 ng/ml in step a).

12. The method according to claim 1 wherein the GSK3 inhibitor is Chir99021, at a concentration of 5 to 10 μM in step b).

13. The method according to claim 1 wherein the GSK3 inhibitor is Chir99021, at a concentration of 6 μM in step b).

14. The method according to claim 1, wherein the BMP4 is used at a concentration of 25 ng/ml in step b).

15. The method according to claim 1, wherein the VEGF is used at a concentration of 200 ng/ml in step c).

16. The method according to claim 1, wherein the forskolin is used at a concentration of 2 μM, in step c).

17. The method according to claim 1, wherein on D0, the pluripotent stem cells are seeded at a density of 50 000 cells/cm.sup.2.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0048] Other characteristics, details and advantages of the invention will emerge on reading the appended figures.

[0049] FIG. 1

[0050] FIG. 1 represents the characterization of the reference endothelial cells on P5.

[0051] (A) Morphology of the HUVEC, HDMEC and CDI iEC reference endothelial cells. (B) Transcriptomic profile of the reference cells, qPCR analysis of the endothelial cell-specific markers PECAM1 (or CD31), CD34, KDR, VE-cadherin (or CD144) and vWF. (C) Protein profile, analysis by FACS of CD31/CD34 and CD31/CD144 and by immunofluorescence of CD31, VE-cadherin (or CD144) and vWF. a) HUVEC, b) HDMEC, c) CDI iEC. (D) Functionality: formation of tubules on GFR-Matrigel, Ac-LDL endocytosis, response to inflammation by treatment with TNFα and observation of ICAM expression.

[0052] FIG. 2

[0053] FIG. 2 represents the method according to the invention and the characterization of the endothelial cells obtained.

[0054] (A) Validated clinical protocol. (B) CD144-APC FACS on D6 (before selection). (C) FACS during the passages for the analysis of the CD31/CD144 and CD31/CD34 co-labelings with the results at P2 representative of the various passages. (D) Functionality of the cells produced during the passages and formation of tubules on GFR-Matrigel.

EXAMPLE 1: CHARACTERIZATION OF THE REFERENCE ENDOTHELIAL CELLS

[0055] HUVEC (Human Umbilical Vein Endothelial Cells) and HDMEC (Human Dermal Microvascular Endothelial Cells) primary endothelial cells and also iPS-derived endothelial cells produced by Cellular Dynamics International (CDI iEC) were used as reference cells for carrying out all the setting up and validation of the quality controls for the iPS-derived endothelial cells: transcriptomic profile (q PCR), protein profile (FACS, immunofluorescence) and functionalities (formation of tubules on Matrigel, response to inflammation, endocytosis capacity).

[0056] The markers analyzed in order to characterize the endothelial cells are CD31 encoded by the PECAM1 gene and CD144 encoded by the VE-cadherin gene, two membrane proteins involved in the intercellular junctions between endothelial cells and which enable endothelium integrity. The vWF cytoplasmic marker is involved in the platelet recruitment during a vessel lesion and is present in mature endothelial cells. It thus makes it possible to have an idea of the maturity of the cells. The CD34 protein is a marker common to endothelial and hematopoietic cells since it is expressed from the hemangioblast stage, said hemangioblast being the precursor of the two cell types. The final marker analyzed is VEGFR2 encoded by KDR, which is the VEGF receptor that itself is involved in angiogenesis stimulation and endothelial cell survival.

[0057] FIG. 1A shows that the 3 reference cell types exhibit the same morphology. They have a similar transcriptomic profile except for the vWF marker which shows a weaker maturity of the CDI iECs compared to the HUVEC and HDMEC (FIG. 1). The HUVEC and HDMEC show more than 98% expression of the CD31 and CD144 membrane markers. The CDI iECs show 93.7% expression of said markers. As for the CD34 marker, a double population, one which is positive and one which is negative to the extent of approximately 50%, is observed for the 3 cell types. The immunofluorescence makes it possible to verify that the vWF maturity marker is indeed expressed in the cytoplasm of the cells (FIG. 10 a, b and c).

[0058] To verify the functionality of the cells, their ability to become organized in a “tubule-like” structure is tested on Matrigel for 24 h. The 3 control cell lines form structures in a network (FIG. 1D).

[0059] The metabolic activity of the endothelial cells is tested by means of the test for endocytosis of Ac-LDL coupled to a fluorochrome. The latter is added to the culture medium of the cells for 4 h and then the cells are rinsed. The endocytosed Ac-LDL remains in the cells and can be observed by the presence of fluorescence inside the cells, demonstrating their endocytosis capacity (FIG. 1D).

[0060] The aim of the final test for functionality of the endothelial cells is to show their ability to respond to inflammation via stimulation with TNFα for 24 h. This treatment enables the overexpression of ICAM, an inflammation marker (stimulator of adhesion and transmigration of leukocytes through the endothelial epithelium). Without treatment, no ICAM expression is observed in the cells (not shown), whereas, with treatment, the cells express ICAM in a similar manner between the 3 cell types (FIG. 1D).

[0061] These results show that the reference cells appear to be functional and express the endothelial cell-specific markers. These are therefore good controls for the cells derived from differentiations from pluripotent stem cells.

EXAMPLE 2: OBTAINING OF HPSC-DERIVED ENDOTHELIAL CELLS AND PHENOTYPIC CHARACTERIZATION OF SAID CELLS

[0062] The hiPSC cells are dissociated with accutase and then seeded at 50 000 c/cm.sup.2 on L7 coating in Stempro hESC SFM medium supplemented with 10 ng/ml of FGF2 and Revitacell at 1/100. The following day, on D1, the medium is replaced with mesoderm induction medium: CTS N2B27 medium (1:1 mixture of KO DMEM-F12 and CTS neurobasal medium supplemented with CTS glutamax, CTS N2 and B27) with Chir99021 at 6 μM and BMP4 at 25 ng/ml.

[0063] On D4, the medium is replaced with the endothelial specification medium: CnT-ENDO medium supplemented with 200 ng/ml of VEGF and 2 μM of forskolin. The medium is renewed the following day. On the 6th day of differentiation, the differentiated cells are dissociated with accutase and selected using CD144+ magnetic beads with LS columns sold by Miltenyi Biotec. The positive cells, that is to say the differentiated endothelial cells, are collected and then frozen in CryoStor.

[0064] The cells are then thawed and seeded on a matrix of collagen I at 10 μg/ml at 20 000 c/cm.sup.2 in CnT-ENDO medium supplemented with 50 ng/ml of VEGF. The medium is changed every 2 days and the cells are passaged every 3-4 days.

[0065] All of these steps are shown schematically in FIG. 2A.

[0066] The cells thus obtained by this method were characterized from the phenotypic and functional point of view. The cells obtained by the method of the invention clearly express the CD31, CD144 and CD34 markers (FIG. 2C) and are functional up to P3 (FIG. 2D).

EXAMPLE 3: COMPARISON OF VARIOUS CULTURE MEDIA AND OF VARIOUS COATINGS

[0067] The protocol of example 2 was carried out using various culture media and various coatings, as indicated in table 1 below.

TABLE-US-00001 TABLE 1 Meso- Endo- % Ex- derm thelial CD144+ peri- induc- specifica- before ment iPS medium Coating tion tion selection CR1 iPS Brew XF GFR- N2B27 SP34SFM 10.5 Stempro hES SFM Matrigel 28.5 iPS Brew XF GMP 5.01 C1 Stempro hES SFM L7 N2B27 SP34SFM 14.9 CTS HSC 21.8 Cnt- 30.5 ENDO iPS Brew XF GMP L7 N2B27 SP34SFM 21.2 CTS HSC 19.7 Cnt- 32.9 ENDO

[0068] The yields obtained (in % of CD144-positive (or CD144+) cells at the outcome of the differentiation step) were compared.

[0069] It results from these tests that the Stempro hES SFM and iPS Brew XF GMP media are both efficient as pluripotent cell culture medium.

[0070] The L7 coating, which is of clinical grade, is as efficient as the GFR-Matrigel coating.

[0071] For the mesoderm induction step, the CnT-ENDO medium is more efficient than the other media tested.